Compositions to effect neuronal growth

ABSTRACT

Compositions containing neurogenic agents for inhibition of neuron death and inducing proliferation of neural cells are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/744,220 filed Jan.17, 2013, now allowed, which is a divisional of U.S. Ser. No. 13/269,507filed Oct. 7, 2011, now U.S. Pat. No. 8,362,262, which is a divisionalof U.S. Ser. No. 12/939,897 filed Nov. 4, 2010, now U.S. Pat. No.8,058,434, which is a divisional of U.S. Ser. No. 12/500,073 filed Jul.9, 2009, now U.S. Pat. No. 7,858,628, which is a continuation of U.S.Ser. No. 12/049,922 filed Mar. 17, 2008, now U.S. Pat. No. 7,560,553,which is a divisional of U.S. Ser. No. 10/914,460 filed Aug. 9, 2004,now abandoned, which claims benefit under 35 U.S.C. §119(e) to U.S. Ser.No. 60/493,674 filed Aug. 8, 2003. The contents of these applicationsare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the use of fused imidazoles, aminopyrimidines,nicotinamides, aminomethylphenoxypiperidines and aryloxypiperidines foruse as therapeutic agents and analytical reagents. More particularlythis invention relates to these agents as therapeutics for preventionand treatment of neurological conditions and diseases in mammals andreagents for detecting neurogenesis and proliferation.

BACKGROUND

According to a long-held doctrine, no significant numbers of neurons aremade in the adult mammal to contribute to the function of the adultmammalian nervous system. However, recent data indicate that adultmammalian brains contain neural precursor cells capable of generatingnew neurons both in normal and in injured conditions. These new neuronshave been quantified in live animals by injecting or feeding in drinkingwater a marker of dividing cells, bromodeoxyuridine (BrdU) and byimmunostaining of post-mortem brains with antibodies against BrdU andneuronal markers. An endogenous marker of dividing cells, Ki-67 protein,has also been used instead of BrdU for this purpose. Thus, in healthy,young rodents, approximately 3,000-15,000 new cells per day areestimated to be born in the dentate gyrus of the hippocampus, about 60%of which express early neuron-specific proteins such as doublecortin andtype III beta-tubulin. Significant numbers of new cells and new neuronshave also been observed in healthy, young primates. In rodents as wellas in primates, the location of neurogenic areas in the central nervoussystem (CNS) is limited to the dentate gyrus of the hippocampus and thesubependymal layer of the striatum. In human patients of different ageswho have been diagnosed with a tumor of the tongue, a single injectionof BrdU has revealed significant number of new cells and new neuronsbeing born in the dentate gyrus and the subependymal layer of thestriatum. Thus, adult mammalian brains contain neural stem cells capableof differentiating into neurons, and this process of generating newneurons (neurogenesis) occurs in the mature, adult brain in significantquantities throughout rodents, primates, and human species.

Such significant quantities of new neurons suggest that the new neuronsmay be important for the normal physiology of the brain, especially thehippocampus. The hippocampus is the main area of neurogenesis in adultrodents and is central for key cognitive functions such as learning andmemory where new information is added, edited, stored, and recalledconstantly throughout life.

Since the hippocampus is the most potent neurogenic area of the brain,many studies have been undertaken to establish whether neurogenesis maybe the cellular mechanism to structurally accommodate theever-increasing volume of cognitive processing to be handled. Thus, ithas been shown that at least some of the newly born neurons, marked bygenetic markers, mature to be electrophysiologically active andintegrate into the existing neuronal circuitry of the hippocampus.Ablation of the neurogenesis in rats leads to decreased cognitivecapabilities in several behavior tests. Thus, the existing datademonstrate that neurogenesis significantly contributes to the normalhippocampal physiology.

For example, most antidepressants are thought to work by increasing thelevels of monoamines available for post-synaptic receptors. Examples ofclasses of agents working apparently by the “monoaminergic hypothesis ofexpression” include the selective serotonin uptake inhibitors (SSRIs)like fluoxetine, the mixed noradrenaline/serotonin transporter blockerslike tricyclic agent imipramine and noradrenaline uptake inhibitors likedesipramine. The antidepressant-induced increase in intraneuronalbiogenic amines occurs quite rapidly. However, theantidepressant-induced improvement in clinical behavior requires weeksof daily administration.

One hypothesis that may account for the slow-onset of theantidepressants' therapeutic activity is that they work by promotinghippocampal neurogenesis. It is expected that neurogenesis would requirea number of weeks for stem cells to divide, differentiate, migrate andestablish connections with post-synaptic neurons. The neurogenesistheory of depression then postulates that antidepressant effect isbrought about by structural changes in the hippocampal circuitrycontributed by newly generated neurons stimulated by antidepressants(Malberg et al., 2000; Czeh et al, 2001; Santarelli et al, 2003).

The neurogenic theory of depression, though not conclusive, has strongsupportive data including the finding that neurogenesis is actuallyrequisite for antidepressant behavioral improvement in the noveltysuppressed feeding model (Santarelli et al., 2003). A therapeuticbenefit from hippocampal neurogenesis is further supported by thefinding of hippocampal atrophy in depression, where magnetic resonanceimaging studies identified a reduction in the right and the lefthippocampal volumes in individuals with major depression (Sheline etal., 1996; Bremner et al., 2000; Mervaola et al., 2000). Long-standingwork also suggests a strong relationship between glucocorticoiddysregulation or glucocorticoid hypersecretion in stress and depression,such that the hippocampal volume loss might be considered a consequenceof glucocorticoid-induced hippocampal neuronal loss (Sheline et al.,1996; Lucassen et al., 2001; Lee et al., 2002 (review)). Furthermore, instudies which involved the administration of a chronic stress toanimals, both hippocampal volume changes and reduction in neurogenesisare observed, and these events are both reversed by chronicantidepressant administration (Czeh et al., 2001; Pham et al., 2003),further illustrating the strong association between depression, stressand neurogenesis. The association comes full circle, since agents orconditions that promote a reduction in neurogenesis also appear ascausative agents/events in depression, specifically glucocorticoid(Sapolsky, 2000), and depletion of serotonin (Brezun and Daszuta, 1999).Kempermann and Kronenberg (2003), though acknowledging the validity ofthe hippocampal neurogenesis theory of depression, suggest that thishypothesis needs to be looked at in the context of a larger model ofcellular plasticity, which elucidates how antidepressants induce nascentneurons of unknown phenotype to survive and produce viable circuits.

Neurogenesis can be characterized as three successive stages:proliferation of endogenous stem cells and precursors, differentiationinto neurons and neuron maturation with formation of viable synapticconnections (plasticity). In consideration of each of these stages ofneurogenesis, the hippocampal volume loss in depression couldpotentially be caused by 1) inhibition of the endogenous hippocampalstem cell proliferation in the dentate gyrus, 2) inhibition ofdifferentiate and dendrite development and 3) loss of neurons(apoptosis) and their dendritic structure. Though apoptosis, also knownas programmed cell death, is observed in depression, hippocampalapoptosis, as measured by DNA fragmentation, from depressed patientsappears to play only a minor role in the volume loss (Lucassen et al.,2001).

In an animal model of acute stress or in normal animals receivingexogenous corticosterone, the stress causes a reduction in synapticplasticity in the hippocampus (Xu et al., 1998). Chromic administrationof the tricyclic antidepressant imipramine, partially reversed the lossin long-term potentiation (LTP) in socially stressed, depressive-likeanimals (Von Frijtag et al., 2001) suggesting imipramine affects theplasticity phase of neurogenesis.

In another animal model of depression characterized by loss ofneurogenesis and hippocampal volume loss, stressed animals thatchronically receive the antidepressant tianeptine, show similar numbersof dividing cells as control animals (no social stress) a measure ofproliferation (Czeh et al., 2001).

In an experiment looking at association of antidepressants andneurogenesis in normal adult rats, the antidepressant, fluoxetine,required chronic administration to cause proliferation of cells indentate gyrus (24 hrs post-treatment), but there was considerable lossof nascent cells, whether in the presence or absence of fluoxetinetreatment, where fluoxetine provided no observed differentiation orsurvival benefit (Malberg et al., 2000).

Results on different neurogenic intervention points by knownantidepressants suggest that novel neurogenic agents that intervene atdifferent points in the neurogenesis pathway could result in potentiallydiverse therapeutic effects on depression. These points of interventioncan be studied and the target elucidated for novel antidepressantcandidates through in vitro assays. Since adult stem cell proliferationand neurogenesis is observed in adult vertebrates in hippocampal dentategyrus (Gould el al., 2001; Eriksson et al., 1998), we can usemulti-potential hippocampal stem cells screen agents in vitro forneurogenic activity.

Interestingly, chronic administration of either the antidepressantfluoxetine, an SSRI or the antidepressant rolipram, a phosphodiesteraseIV inhibitor, promoted neurogenesis in normal animals (Malberg et al.,2000; Nakagawa et al., 2002). One might conclude from these results thatany agent that promotes neurogenesis will generate a behavioral benefitin depression, unrelated to the agents' mechanism-of-action or possiblythat there is a common pathway where both drug actions overlap. D'Sa andDuman suggest a scheme whereby the phosphorylation and activation ofCREB and the subsequent expression of BDNF are central to the inductionof neurogenesis that culminates in antidepressant behavior. CREBphosphorylation is increased in animals administered rolipramchronically (Nakagawa et al., 2002), and antidepressants that eitherincrease Ca²⁺/CaM-kinases or cAMP could cause the phosphorylation ofCREB in the nucleus (reviewed by D'sa and Duman 2002). D'Sa and Duman(2002) further suggest that the phosphorylated CREB then binds to theCRE binding site to promote the expression of BDNF and bcl-2, thatappear critical to cell survival and plasticity. Proof of involvement ofneurotrophic factor BDNF in depression comes from studies showing thatantidepressants and electroconvulsive shock both caused an increase inBDNF levels (Nibuya et al, 1996) and that intrahippocampal injection ofBDNF had antidepressant activity in two models of depression (Shirayamaet al., 2002).

If neurogenesis is critical for antidepressant activity is it alsosufficient for therapeutic activity and is the mechanism by which theneurogenesis occurs or timing of neurogenesis also critical totherapeutic activity? These questions can be answered by using novelagents developed through screening paradigms that identify agents thatpromote the proliferation and differentiation of endogenous hippocampalstem cells to neurons in vivo and determine if they will be effectiveantidepressants.

In abnormal conditions, such as when an injury to a brain area hasoccurred, neurogenesis becomes more wide-spread and perhaps functionallydiverse. In rodent models of ischemic and hemorrhagic stroke, the newlyborn neurons of the subependyma (also referred to as subventricularzone) are seen migrating to and accumulating in the lesion area of thecortex. However, the newly born neurons have a short survival period.

Neuropathology associated with key cognitive regions of the Alzheimer'sdiseased brain has led to therapeutic strategies that address theneuronal loss, in the hopes of reducing the cognitive decline. Onestrategy enlists trophic agents, that regulate neuronal function andsurvival, as Alzheimer's Disease (AD) therapeutics (see Peterson andGage, 1999). Problems with systemic administration, side effects andlocating trophic-sensitive neurons generated few clinical successes withthese therapies. One AD therapeutic, AIT-082, promotes memoryenhancement in AD individuals potentially by stimulating endogenoustrophic factors (Ritzman and Glasky, 1999; Rathbone et al., 1999). Sothe use of agents to promote increased survival and function of theremaining available neurons appears to have some therapeutic value.

As discussed, the hippocampus is one of the main brain regions whereneurogenesis in adult brain has been documented across severalvertebrate species, including monkeys and humans (e.g., Gould et al.,2001; Eriksson et al., 1998). In fact, adult hippocampal neurogenesiscontributes functionally to cognitive capacity. Shors et al, (2001)reported that inhibition of neurogenesis in adult rat hippocampus, inthe absence of the destruction of existing neurons, caused impairedmemory function. Many studies observed that degenerative conditionsinduced, neurogenesis in mature mammalian brains, suggesting theexistence of a natural repair pathway by means of neurogenesis. A focalischemic model, reversible photothrombic ring stroke, caused increasedneurogenesis in rat cortex by 3-6% (Gu et al., 2000). Seizures inducedby electroconvulsive shock in adult rats increased neurogenesis indentate gyrus of hippocampus (Scott et al, 2000; Madsen et al, 2000).Also, rats gamma-irradiated to kill mitotic cells in the CNS showedreduced numbers of nascent neurons and reduced LTP in slice cultures.These observations highlight the likelihood that a cellular mechanismfor neurogenesis within adult human CNS, especially in hippocampus, doesexist both as a normal physiological process and as a self-repairingpathway.

In adult neurogenesis a decline due to aging is observed (Kuhn et al.,1996), though proof that this age-dependent decline in neurogenesiscauses cognitive impairment is still debated. Considerable evidence doesexist, indicating that increased neurogenesis reduces age-associatedcognitive decline. This is most dramatically observed with thetransplantation of human stem cells into aged rats initiating improvedwater maze learning and retention (Qu et al., 2001). Other data suggeststhat induction of neurogenesis by diet restriction (Lee et al., 2000)exercise (van Prang et al., 1999) or growth factor addition(Lichtenwalner et al., 2001) improves learning and memory in adult oraged rats. A number of other inducers of neurogenesis have beenidentified, including anti-depressants (Malberg et al., 2000; Czeh etal., 2001), and nitric oxide donors (Zhang et al., 2001) suggesting theusefulness of neurogenic agents for other diseases presentingcognitive-deficits, such as stroke and depression. A small molecule thatinduces hippocampal neurogenesis that is blood brain barrier penetrablewould allow for a potentially novel oral therapeutic for Alzheimer'sdisease.

Other potential AD therapeutics progressing in clinical trials, targetneurodegeneration in the hopes of reducing the neuronal loss andcognitive decline. Apoptotic death involving caspase pathways and DNAfragmentation has been measured in in vitro and animal models of AD andin Alzheimer's diseased brain tissue. The extent of apoptosis leading toneuronal loss is of continual debate with most agreeing it has someeffect, but that other neuronal death pathways definitely play a role(see Behl, 2000; Broe et al., 2001, Roth, 2001). Concern that measuresof upstream caspase markers in neurons from AD tissue may not proceed todegeneration has been suggested (Raina et al, 2001). In order to screenfor a neuroprotectant therapeutics it is critical, therefore, to measuremore than one endpoint of neuronal death and determine at what point anagent may intervene in the death pathway(s). Behl (2000) suggested thatAD pathology is most likely a mixture of apoptotic and necrotic pathwaysand that concentrating therapeutic discovery using only one pathway mayprovide inconclusive results. All hits in our neurogenesis models weretested through our secondary apoptosis/necrosis assay to screen foragents that function both as neurogenic and neuroprotective agents.These agents may improve or reverse the cognitive decline observed inMCI and AD.

Therefore, recent studies indicate that neurogenesis occurs in the adulthuman brains under normal as well as under degenerative conditions andthat such adult-generated neurons do contribute functionally to thebrain physiology such as learning and memory. These observationshighlight the likelihood that a cellular mechanism for neurogenesiswithin adult human CNS, especially in hippocampus, does exist both as anormal physiological pathway and as a self-repairing pathway. What isnot known is whether deficiencies in the volume or persistence ofneurogenesis and/or the survival or maturation of the new neuronscontribute to permanent damage.

Thus, a compound that can stimulate endogenous neurogenesis, either in adisease state or in a healthy state, may be an effective drug for anumber of human nervous system conditions and diseases. Manyneurological diseases, including Alzheimer's disease, mild cognitiveimpairment, dementia, age-related cognitive decline, stroke, traumaticbrain injury, spinal cord injury, and the like, are neurodegenerativeconditions. Neuropsychiatric diseases including depression, anxiety,schizophrenia and the like also show nerve cell dysfunction leading tocognitive, behavioral, and mood disorders. A neurogenic drug or agentthat enhances the process of generating new neurons (neurogenesis) wouldbe beneficial for countering and treating these diseases.

Candidate drugs generated from the screening have been tested in variousanimal models of human neurological and psychiatric disorders todetermine the drugs' therapeutic potentials. An effective, predictive invitro assay that can be used to select for clinical drug developmentneurogenic compounds that is particularly effective in promoting theneurogenesis in vivo has been described in U.S. patent application Ser.No. 10/728,652 filed Dec. 5, 2003, which is incorporated herein byreference in its entirety.

SUMMARY

The present invention relates to compounds that promote neurogenesis invivo. More particularly, the present invention is related to classes ofcompound structures that are shown to be particularly effective inpromoting neurogenesis including compounds of the type, fusedimidazoles, aminopyrimidines, nicotinamides, aminomethylphenoxypiperidines and aryloxypiperidines. These compounds are shown topromote neurogenesis by proliferation and/or differentiation of humanhippocampal multipotent stem cells and/or progenitor cells and neuronalprogenitors. Moreover, the present invention relates to these agents astherapeutics for prevention and treatment of neurological diseases inmammals and as reagents for detecting neurogenesis and proliferation.

In one embodiment of the present invention, compounds are evaluated fortheir ability to promote neurogenesis by proliferation/differentiationof human hippocampal multipotential stem cells and neuronal progenitorsand whether small molecule agents of the above chemical structures thathave neurogenic activity in vitro and/or in vivo also have the abilityto inhibit neuronal death. Modulation of neurogenesis pathways mayoverlap pathways critical to apoptotic and necrotic neurodegeneration,therefore, neurogenic small molecule agents are tested for their abilityto inhibit neurodegeneration.

The compounds of the present invention are shown to stimulate endogenousneural stem cells capable of differentiating into neurons in adult humanbrains to proliferate and to differentiate into functional neurons invivo. The additional neurons may enhance the cognitive ability of thesubject and significantly extend the ability to perform cognitive tasksduring extended periods of sleep deprivation.

The present invention also includes the identification of a specificclass of compounds, the aminopyrimidines, that appear especiallyeffective at inhibiting apoptosis in various neurodegenerative modelssuggesting this class of compounds might be especially useful forneurodegenerative indications.

The present invention includes neurogenic drugs which serve to preventor treat neurodegenerative and neuropsychiatric disorders by promotingthe endogenous birth of new neurons within the nervous system byadministering the compounds of the present invention to the patientalone or in combination with transplanted stem cells or progenitorcells.

In a further embodiment of the present invention, the compounds are usedas reagents for detecting neurogenesis and proliferation in an in vitroassay.

In one embodiment of the present invention, an agent is administered totreat a neurodegenerative disease. In a preferred embodiment of thisinvention the neurodegenerative disease includes Alzheimer's disease,dementia, mild cognitive impairment, aged-related cognitive decline,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,demyelination, stroke, spinal injuries, traumatic brain injuries,neuropathic pain, and the like.

One embodiment of the present invention includes an agent administeredto treat a psychiatric disease. In one embodiment, the psychiatricdisease includes depression, postraumatic stress syndrome, acute orchronic stress, anxiety, schizophrenia, sleep deprivation, cognitivedysfunction, amnesia, and the like.

One embodiment of the present invention includes an agent administeredby any number of routes including combining with multipotent stem cellsor differentiated multipotent stem cells transplanted into brain.

In yet another embodiment of the present invention, the agent isadministered to treat a cognitive dysfunction, memory deficit, ordecreased learning capacity.

In a further embodiment of the present invention, cognitive dysfunctionincludes sleep deprivation, moderate cognitive impairment (MCI), and thelike.

In another embodiment of the present invention, the agent isadministered to enhance cognitive function, memory, and/or learningcapacity of an individual.

In another embodiment of the present invention, an agent is administeredby at least one route and at least one multipotent stem cell ordifferentiated multipotent stem cell is transplanted into brain.

In another aspect of the present invention, the agent is utilized in theabove methods. Additional features and advantages of the presentinvention are described in, and will be apparent from, the followingDetailed Description of the invention and the figures.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph illustrating an example of a proliferation profileof compounds selected from primary screening. Proliferation is measuredafter compound treatment for seven days by Alamar Blue staining of livecells per well. Shown are relative values over the vehicle control.

FIG. 2 is a bar graph illustrating an example of a neurogenesis profileof compounds selected from primary screening. After seven days ofcompound treatment, the cells were stained with the neuron-specificanti-tubulin IIIb antibody. TuJ1, for neurons and Hoechst for all cellnuclei. Shown are the relative ratio of neuron: total cells for eachcompound over the vehicle control in percentage. Typical ratio forvehicle control is 40-50% neurons. The ratio can change by eitherincreased differentiation of stem cells to neurons, decreasedproliferation of astrocytes, or increased proliferation of neuronalprogenitors.

FIG. 3 is a bar graph illustrating an example of a neurogenesis profileof compounds selected from primary screening. After seven days ofcompound treatment, the cells were stained with TuJ1 for neurons. Thenumber of TuJ1+ neurons per area was quantified and expressed as arelative value to the vehicle treated control.

FIG. 4 is a line graph illustrating an example of a neurogenesisdose-response profile of selected compounds. Differentiating humanhippocampal stem cells were treated for seven days with varyingconcentrations of “primary hits”. Subsequently, the cells were fixed,stained with anti-beta tubulin antibody, and positive cells werequantified by Array Scan II. Shown are the number of neurons after eachtreatment normalized against the no-compound control.

FIG. 5 is a bar graph illustrating an example of results of an assay foran in vitro model of neuroinflammation. Human hippocampal stem cellswere differentiated for four weeks and then treated with the spentmedium collected from human monocytes pre-treated for twenty-four hourswith either the vehicle alone (RPMI medium) or with 0.5 ug/mllipopolysaccharide (LPS). Lactate dehydrogenese (LDH), a number ofneuroinflammation, in the neuronal culture media was measuredtwenty-four hours post incubation of the hippocampal cells with themonocyte supernatants.

FIG. 6 is a bar graph illustrating an example of a neuroprotectionprofile of the compounds in the neuroinflammation assay. Humanhippocampal stem cells were differentiated for four weeks and thentreated with the spent medium collected from human monocytes preheatedfor 24 hours with either the vehicle alone or with 0.5 ug/mllipolysaccharide (LPS). LDH in the neuronal media was measured at 24hours post incubation of the hippocampal cells with monocyte supernatantwhile the number of neurons indicated by MAP-2 staining was measuredafter forty-eight hours of treatment.

FIG. 7A is a bar graph illustrating an example of a neuroprotectionprofile of the compounds against staurosporine-induced human hippocampalneurodegeneration as indicated by loss of neurons after twenty-four hourtreatment with staurosporine by counting the remaining number ofneurons, an increase in unclear fragmentation, and an increase inintracellular levels of activated caspase-3/-7 detected byimmunostaining. Addition of 125 nM staurosporine solution to humanhippocampal stem cells differentiated for 3-4 weeks causes neuronalinjury and death. Effect of the competed treatment to inhibit thestaurosporine-induced neuronal loss and injury are shown. Each barrepresents n=6 wells of the mean+/−the SEM from a single experiment.

FIG. 7B is a bar graph illustrating an example of a neuroprotectionprofile of the compounds against staurosporine-induced human hippocampalneurodegeneration as indicated by an increase in nuclear fragmentationafter twenty-four hour treatment with 125 nM staurosporine solution tohuman hippocampal stem cells differentiated for 3-4 weeks. Effect of thecompound treatment to inhibit the staurosporine-induced neuronal lossand injury are shown. Each bar represents n=6 wells of the mean+/−theSEM from a single experiment.

FIG. 7C is a bar graph illustrating an example of a neuroprotectionprofile of the compounds against staurosporine-induced human hippocampalneurodegeneration as indicated by an increase in intracellular levels ofactivated caspase-3/-7 detected by immunostaining after twenty-four hourtreatment with 125 nM staurosporine solution to human hippocampal stemcells differentiated for 3-4 weeks. Effect of the compound treatment toinhibit the staurosporine-induced neuronal loss and injury are shown.Each bar represents n=6 wells of the mean+/−the SEM from a singleexperiment.

FIG. 8 is a bar graph illustrating an example of a neuroprotectionprofile of the compounds against apoptosis, also known as programmedcell death, induced by beta amyloid peptide 25-35 as a model ofAlzheimer's disease. Caspase-3 activation plays a central role inapoptosis and is an indicator of the occurrence of apoptosis ifdetected. Shown are three agents, NSI-182, NSI-144 and NSI-130, thatinhibit apoptosis induced by beta amyloid peptide 25-35. Bars representmean±SD from five wells.

FIG. 9A and FIG. 9B are micrographs illustrating an example of BrdUimmunostaining of a mouse dentate gyrus having been administered avehicle and a compound. Mice were treated daily with various testcompounds at 10 mg/kg p.o. (FIG. 9B) or vehicle alone (FIG. 9A) for tendays. The animals were injected daily with BrdU for the first sevendays. At the end of the ten-day period, the animals were perfused andtheir brains sliced for immunostaining with anti-BrdU antibody andanalysis. FIG. 9B illustrates that the NSI-158 treated brain has fargreater dividing cells in the dentate gyrus of the hippocampus thanvehicle treated mouse as illustrated in FIG. 9A.

FIGS. 10A and 10B are bar graphs illustrating an example ofquantification of BrdU-immunopositive cells in the in vivo neurogenesistesting the compounds. Mice were treated with the various test compoundsfor in vivo neurogenesis in two cohorts. The post-mortem cell counts ofBrdU-immunopositive cells of the two cohorts are shown in FIGS. 10A and10B. The cells from one dentate gyrus of each mouse in each test groupwere counted and average BrdU values immunoreactivity are shown. Thesymbols, * and ** indicate immunoreactivity increases of statisticalsignificant the indicated p values. Seven compounds are especiallyeffective in increasing the neurogenesis in the dentate gyrus of thehealthy adult mice, the area known to have continued adult neurogenesis.

FIG. 11 is a bar graph illustrating an example of in vitro neurogenesisprofile of the compounds for a long-term treatment. Differentiatinghippocampal human stem cells mere treated with vehicle alone or with thetest compounds for three weeks. Increase in cell number was determinedcompared to the control. Shown are several examples that are effectivein continued neurogenesis. Bars represent mean+/−SD from six wells.

FIG. 12 is a bar graph illustrating an example of differentiation intomature neurons as a percentage of control in response to a three-weektreatment with the test compounds. Mitotic cells were labeled with BrdU.BrdU-positive cells were double-stained with the marker of matureneurons, anti-MAP-2 antibody. Total BrdU-immunostained cells andBrdU+/MAP-2ab+ co-stained cells indicating neurons were counted.

FIG. 13 is a bar graph illustrating an example of neuronalidentification of the mitotic cells in response to the compoundtreatment. Mitotic cells were labeled with BrdU during the lastforty-eight hours of the two-week treatment with the test compounds.BrdU alone and BrdU together with TuJ1 as co-stained cells werequantified. Results from NSI-106 and LIF treatment are shown as anexample. Unlike with LIF, NSI-106 produces mostly co-stained cells,indicating that the mitotic population is committed neuronalprogenitors. The bars represent the mean±SD from each of six wells.

FIG. 14 is a micrograph illustrating an example of production ofcommitted neuronal progenitors by NSI-106. Differentiating hippocampalstem cells were treated with NSI-106 for 10 days, fixed, and stained forKi-67 protein as well as TuJ1. Ki-67 protein is an endogenous marker ofmitosis. Significant number of cells were co-stained suggesting thatNSI-106 not only increase the number of dividing stem cells but causesincrease in dividing neuronal precursors or committed neuronalprogenitors as indicated by Ki-67 positive and TuJ1 positive cellsdemonstrating neuronal morphology.

DETAILED DESCRIPTION

The present invention relates to the identification of a specific typeof compounds including fused imidazoles, aminopyrimidines,nicotinamides, aminomethyl phenoxypiperidines and aryloxypiperidines foruse as therapeutic agents and analytical reagents by means of promotingneurogenesis by proliferation/differentiation of human hippocampalmultipotent stem/progenitor cells and neuronal progenitors. Moreparticularly this invention relates to these agents as therapeutics forprevention and treatment of neurological diseases in mammals andreagents for detecting neurogenesis and proliferation.

The key activity of a neurogenic agent is to increase the number ofneurons generated from their precursors. A neurogenic agent can bringabout such an increase in the number of neurons by a number of differentmechanisms. The neurogenic agent can increase the number of neuronsgenerated from their precursors (neurogenesis) and/or can protectexisting neurons from neurodegeneration (neuroprotection).

The neurogenic agent can increase neurogenesis by acting as a mitogenfor the neural stem/progenitor cells and, thereby, increasing theprogenitor's cell number which, in turn, results in increased number ofneurons in the culture when differentiated. Another mechanism includesthe neurogenic agent acting as a neuronal specification factor bypromoting the stem/progenitor cell differentiation toward neurons at theexpense of glia. This directed differentiation will also result inincreased number of neurons in the culture, but without changing theoverall cell number. In yet another mechanism, the neurogenic agent canact as a mitogen for committed neuronal progenitors that differentiateonly into neurons. Increasing this subpopulation would also increase thefinal number of neurons in the culture. Alternatively the neurogenicagent can act as a neuroprotectant to decrease neurodegeneration byacting as a survival factor to rescue immature neurons from undergoingcell death during differentiation, which will result in increasedneurons.

Initial compound libraries include directed-libraries based onmechanistic pathways or targets thought to be involved in mitosis,differentiation, and survival of neural cells. These targets includegrowth factor receptors (e.g., FGFR, EGFR, NGFR), signal transductionpathways such as ras, CREBP, protein kinases and phosphatases,cell-cycle regulators such as c-myc, p53, p21, transcription factorssuch as bHLH proteins and nuclear hormone receptors, extracellularmembrane (ECM) proteins such as metalloproteinases, and stress-relatedfactors. These targets represent a collection of non-overlappingpharmacophores which cover diverse chemical space and, at the same lime,lead to rapid identification of structure-activity relationships.

A neurogenesis screen of components to discover a safe drug which canactivate the stem cells within the brain of a subject, especially in thehippocampus, and which can recruit new neurons into the circuitry tobroaden the neural functionality at the physiological level must becapable of identifying a compound that will significantly boost eitherof these processes. In vitro assays in 96-well format have been designedto detect the effect of a compound on the proliferation anddifferentiation of hippocampal stem cells. These assays have been usedto screen compound libraries for activities that stimulate mitosisand/or differentiation of hippocampal stem cells and/or progenitors.Candidate drugs generated from the screening will be tested in variousanimal models of human neurological and psychiatric disorders todetermine the drugs' therapeutic potentials.

The compounds identified as potential neurogenic agents includecompounds with the following general structures:

Structure Formula 1: Fused Imidazoles

Structure Formula 2: Aminopyrimidines

Structure Formula 3: Nicotinamides

Structure Formula 4: Aminomethyl Phenoxypiperidines

Structure Formula 5: Aryloxypiperidines

Culturing a Stable Cell Line of Neural Progenitors

A screening of a large number of unknown agents (e.g., protein factors,peptides, nucleic acids, natural compounds, or synthetic compounds) fordiscovering a candidate drug involves repeating the same test forhundreds to millions of times. This repeated testing requires high levelof reproducibility from the test. In order to obtain suchreproducibility for a neurogenesis assay, stable cell lines of neuralprogenitors are required which, upon differentiation, generatereproducible quantities of neurons. For this purpose, a cell line isdefined as a population of cells having been expanded for at least tencell-doublings.

Human and other mammalian neural stem cells have been isolated,expanded, and differentiated, in culture from all major areas of thebrain and spinal cord. (See for example, U.S. Pat. Nos. 5,753,506 and6,040,180). Hundreds of stable, characterized, and cryopreserved neuralstem cell lines have been isolated from many areas of the human androdent brains including the hippocampus. In one embodiment, amultipotent neural stem cell/progenitor cell line derived from humanhippocampus is used. As discussed above, the hippocampus is known forits relatively high level of neurogenesis. Cell lines derived from otherareas of the central nervous system (CNS), including the dentate gyrusof an adult brain, can also substitute for the hippocampus. A neuralprogenitor population derived as a stable cell line from partialdifferentiation of embryonic stem cells can also be used.

Although cell lines that are not genetically engineered are preferred,in one embodiment, cell lines that are genetically engineered to enhancethe mitotic capacity of the cells are used to test potential neurogenicagents. In one embodiment, the genetic modification consists ofintracellular over-expression of functional c-myc protein under aconditional activation system such as a c-myc protein fused to aligand-binding domain of an estrogen receptor as described in U.S.patent application Ser. No. 09/398,897 and filed Sep. 20, 1999, andincorporated herein by reference.

In one embodiment, a progenitor population that, upon differentiation,generates both neurons and glia in a single culture has been used.Presence of glia, either astrocytes and/or oligodendrocytes, or theirprecursors, are required to promote physiological maturation of nascentneurons born from their precursors in culture.

In one embodiment, differentiation of the progenitors is initiated bywithdrawing the mitogen from the culture. It is preferable that serum aswell as other growth-promoting factors, are removed from, or not addedto, the differentiating culture due to their significant effect on thereproducibility of the neurogenesis assay.

Detection of Cell Proliferation

Neural stem cells and progenitor cells differentiate spontaneously inthe absence of a mitogen; therefore, undifferentiated mitotic cells areharvested by enzyme treatment to remove residual mitogen, such as basicfibroblast growth factor (bFGF) used in expansion and proliferation ofthe cells. In one embodiment of the present invention, undifferentiatedhuman hippocampal stem cells (e.g., HH580)/progenitor cells areharvested by enzyme treatment.

The collected cells are seeded for attachment of the cells such asstandard 96-well or 384-well plates, pre-coated with extracellularmatrix proteins, such as poly-D-lysine and fibronectin (e.g., BiocoatPDL, Fisher). The initial seeding density can be within the range ofabout 2,000-125,000 cells per well of a 96-well plate. The preferreddensity, however, is 40,000 cells per well of a 96-well plate which hasbeen optimized for best signal-to-noise ratio as described in pendingU.S. patent application Ser. No. 10/728,652. Cell density that is toolow retards the initiation of differentiation and results in poorplating efficiency which interferes with the assay. Cell density that istoo high leads to inhibition of neurogenesis due to cell-to-cellinteraction and paracrine factors which also interfere with the assay.It should be appreciated that the actual cell number can beproportionally decreased or increased depending upon the surface area ofthe culture substrate used. For example, for a 384-well plate which hasapproximately one-fourth of the surface area of a 96-well plate, theinitial seeding density should be decreased by one-fourth accordingly.

The cells are passaged and grown for seven days in 15 cm plates in fullgrowth medium according to Neuralstem protocol with 50% of the mediachanged every other day. Following centrifugation, the call pellet isre-suspended in N₂b medium and enough N₂b medium is added to the cellsuspension to achieve a concentration of 4×10³ cells per ml. The N₂bseeding media includes a standard serum-free, growth factor-free, basalmedia without phenol red that supports healthy neuronal/glial survival.Fibronectin and PBS is completely removed by aspiration from wells offibronectin-coated 96-well plate(s). 100 μl of N₂b medium with orwithout 2× concentration of screening agents or compounds to be testedare added to each well. Subsequently, 100 μl of the cell suspension isadded to each well at a density of 40,000 cells per well. The cellsuspension is incubated for two days at 37° C. in the presence of 5%CO₂.

On Days 2, 4, and 6 of post-plating, incremental additions of thecompounds are made to each well at appropriate concentrations.Therefore, after two days, 100 μl of N₂b medium is removed from thewells, and 100 μl of the N₂b medium is added. Likewise, on days four andsix, 100 μl of N₂b medium is removed from the wells and 100 μl of theN₂b medium is added, and the cell suspension is again incubated at 37°C. in the presence of 5% CO₂.

On day seven, the final day of the culture, a fluorescent dye such asAlamar Blue dye is added to each well. Alamar Blue dye detects metabolicrespiration and is a reflection of total cellular activity indicatingchanges in cell number of neurons. In this regard, 20 μl of Alamar Blue(Biosource International DAL 1025) is added to each well and incubatedfor two hours at 37° C. in the presence of 5% CO₂.

As a preliminary detection of positive activities, the overallimmunostaining intensity in each well is read by a fluorescence platereader. For instance, the fluorescence of the oxidized dye in each wellis read by a fluorescent plate reader such as the Molecular DevicesFluorometric Plate Reader. The reader is set on the Read Mode End Pointsetting at an excitation of 530 nm, an emission of 590 nm, and a cutoff570 nm. The fluorescence level is proportional to the number ofrespiring cells in the culture and is a measure of a proliferativeactivity of a test agent. The data is then exported and saved into anysuitable database such as a Microsoft Excel Workbook

FIG. 1 illustrates the level of proliferation of the cells aftertreatment with a number of compounds described in Tables I and II forseven days by Alamar Blue staining of live cells per well. Themeasurement of proliferation is represented by a relative ratio ofneurons expressed as a percentage of the total cells for each compoundto the total cells for the vehicle control. The typical ratio of neuronsto the vehicle control is 40-50%. The ratio can change by eitherincreased differentiation of stem cells to neurons, decreasedproliferation of astrocytes, or increased proliferation of neuronalprogenitors.

After the Alamar Blue assay to detect general cellular proliferation,further staining with other matters are required to determine what celltypes (e.g. neuronal, glial) are observed under differentiating media.

Detection of Neurogenesis

The final neuron number is detected by immunostaining of the culturewith antibodies against neurons and is quantified by counting of theimmunopositive neurons and/or by measuring the staining intensity.Therefore, after seven days of compound treatment, the cells are stainedwith the neuron-specific anti-tubulin IIIb antibody, TuJ1, for neuronsand Hoechst for all cell nuclei. The number of TuJ1+ neurons per area isquantified and expressed as a value relative to the vehicle-treatedcontrol of Hoechst-stained cells. In this regard, the medium is removedfrom the wells by decanting and blotting the plates on paper towels. Thecells are then fixed in 100 μl of 4% paraformaldehyde per well forthirty minutes at room temperature. The cells are washed three timeswith 400 μl per well of phosphate-buffered saline (PBS) at pH 7.5 forapproximately ten minutes per wash. The plates are then stored at 4° C.in PBS or used immediately for staining.

The cells are subsequently stained with antibodies againstneuron-specific antigens according to standard procedures. The stainingprocess includes decanting the PBS and blocking the cells with 100 μl of5% Normal Goat Serum (NGS) in PBS for one hour at room temperature. TheNGS/PBS is decanted and the plates are blotted on a paper towel. Thecells are then permeabilized with 0.1% Triton X-100 in PBS for 30minutes at room temperature.

Typical neuron-specific antigens include Type III-beta tubulin andMAP-2c. The total cell number in each well was quantified by stainingthe cultures with a nuclear dye such as DAPI or Hoechst according tostandard procedures. Therefore, once the cells are permeabilized, 20 μlof TuJ1, monoclonal antibody against neuronal class 111 β-tubulin(CoVance (BabCo) MMS-435P) and 32 μl of anti-GFAP polyclonal (DAKOZ0334) are added to and mixed of PBS with 5% NGS. Final dilutions ofTuJ1 and anti-GFAP are 1:400 and 1:250, respectively. 90 μl of theantibody solution is added to each well except two eleventh column. 90μl of PBS/5% NGS is added to the eleventh column and no cells are addedto the twelfth column. The plate(s) are sealed with parafilm andincubated at 4° C. overnight.

After incubating the cells overnight, the antibody solution is decantedand 200 μl of PBS/0.1% Triton X-100 is added to the wells. The plate(s)are placed on a shaker and incubated at room temperature with gentleshaking for ten minutes. The cells are again washed with the PBS/TritonX-100 solution another two times.

Following staining with TuJ1 and anti-GFAP, 20 μl of Alexa Fluor 488labeled goat anti-moose IgG (H+L) (Molecular Probes A-11001) and 100 μlof LRSC-IgG, goat anti-rabbit (Jackson Immune 111-295-144) is added andmixed with 10 ml of PBS/5% NGS. 100 μl of the resulting antibodysolution is added to each well. The plate(s) are covered with foil andincubated with gentle shaking for up to one hour at room temperature.The cells are again washed as described above with PBS and an TritonX-100. 200 μl of PBS is then added to each well and microscopy isperformed to detect positive TuJ1 and GFAP staining.

If the cells are not positive for TuJ1 and GFAP staining, one 20 μlaliquot of DAPI (Molecular Probes) or Hoechst (Sigma Chemical) isdiluted in 2 ml of PBS. 20 ul of this solution is then added to eachwell. The plates are left at room temperature for 15 minutes and thenwashed twice with PBS. After a final wash 200 μl of PBS is added to eachwell.

The plate(s) are then read by the Molecular Devices Fluorometric PlateReader to determine the ratio of TuJ1+ cell number (neurons) toHoechst-stained cells (total cells). The reader is set on the Read ModeWell Scan setting. For detection of TuJ1/Alexafluor 488 staining thesettings include an excitation wavelength of 495 nm, emission wavelengthof 519 nm with auto cutoff, and a PMT Sensitivity set on high. Fordetection of GFAP/LRSC staining, settings include an excitationwavelength of 570 nm an emission wavelength of 590 nm with auto cutoff,and a PMT Sensitivity set on medium. For detection of DAPI/Hoechststaining the excitation is set at 358 nm, the emission is set at 461 nmwith auto cutoff, and the PMT Sensitivity is set on high.

FIG. 2 for example illustrates an example of a neurogenesis profile ofcompounds selected from primary screening which provides the relativeratio of neurons expressed as the total cells for each compound as apercentage of the total cells for the vehicle control. The typical ratioof neurons to the vehicle control is 40-50%. To this end, a compoundwith 130% or greater Alamar Blue reading compared to that of the vehiclecontrol is selected as an active compound for the proliferation effect.A compound with 110% or greater increase in the neuronal number or theneuronal proportion over the control as indicated by immunostainingusing other markers is selected as an active agent for the neurogeniceffect. For the positive hits, more quantitative analysis is carried outby automated morphometric counting of individual cells.

Primary Screening of Unknown Compounds

Synthetic organic compounds for screening through the neurogenesisscreen are pre-selected by predicted bioavailability and CNSpermeability. Calculations for CNS permeability is based on the use of300 descriptors for 1,474 known CNS therapeutics. Predicted propertiesfor successful CNS drugs are determined to be: (1) molecular weight (MW)of 400 or less, (2) five or fewer hydrogen bond acceptors, (3) two orfewer hydrogen bond donors, (4) polar area below 120 angstrom, and (5)neutral or basic with a pKa between 1.5 and 10.5.

Since the screening of 10,000 small molecule compounds in in vitromodels of neurogenesis has been completed the in vitro screen have beenshown to be predictive of in vivo neurogenic efficacy, orally availableagents can be tested, that promote in vivo neurogenesis in models ofdepression.

Rolipram, for example, an antidepressant that works by increasing cAMPlevels and is neurogenic in animals (Nakagawa et al., 2002) waseffective in a primary in vitro neurogenesis screen. This suggests thatthe primary in vitro screen includes those agents that might promoteneurogenesis by targeting the cAMP/pCREB/BDNF pathway. This does notnecessarily exclude all other neurogenesis mechanisms for theneurogenetic agents. If the target of these neurogenic agents isimportant for behavioral activity where three separate chemicallydiverse classes showed in vitro assay efficacy differences and that themechanism for all does not overlap at the point of CREB phosphorylationand BDNF expression then one might expect very different effects onbehavioral activities in depression models.

Cumulatively, over five thousand synthetic compounds of the typeincluding fused imidazoles, aminopyrimidines, nicotinamides, aminomethylphenoxypiperidines and aryloxypiperidines have been evaluated for theireffect on neurogenesis according to the assay method described above.From the preliminary analysis using the fluorescent plate reader, overthree hundred compounds show initial positive activity. Those threehundred compounds have been re-analyzed by quantitative neuron counting.Among them, thirty compounds significantly increase cell number orproliferation; fifty-three compounds increase the number of neuronsneurogenesis; and seven demonstrated significant activity in bothproliferation and neurogenesis. The significance level was empiricallyset at an activity above 30% change over the vehicle control forproliferation and above 10% change for neurogenesis.

Sixteen compounds possessing exceptional in vitro neurogenic capacityhave been identified and are listed along with their structures in TableI. The sixteen compounds have been tested for neurogenic activities inlive mice as described below and in in vitro assays. Table II detailsthe efficacy results of the sixteen effective small molecule agents.Based on these findings, the compounds have potentially effectivetherapeutic value for any number of neurodegenerative andneuropsychiatric disorders. These disorders include, but are not limitedto, stroke, Alzheimer's disease, Parkinson's disease, depression, mildcognitive impairment (MCI), traumatic brain injury and the like.

Dose-Response Profiles

In another in vitro assay, compounds are tested as above at varyingconcentrations to obtain a dose-response curve and EC50 values(effective concentration of the compound which produces 50% of themaximum possible response for that compound). Compounds with EC50 valuesbelow 10 μM for neurogenesis or proliferation effect are consideredfurther.

Primary dose-response profiles and secondary functional assays are usedto further eliminate compounds from the primary screen based on a lackof dose-dependent effect on neurogenesis, indicated by lack of linearityof dose-response, and in vitro neurotoxicity. The dose-response curvemeasures neurogenesis over a concentration range of 100 pM to 100 μM.Examples of several primary hits fully analyzed for dose-response areshown in FIG. 4. Significantly, most compounds exhibit a linear responseover several log concentrations below 1 μM. This linear responseindicates that the assay for primary screening is reliable and that thequality of the compound library is high. Table II contains the summaryof EC50 of each compound tested. On the other hand, at highconcentrations such as 100 μM, some, but not all, compounds testedshowed a high level of neurotoxicity, indicating that analyzingdoss-response curves will be discriminatory and serve as an effectiveearly filter.

Detection of Neuroprotection

Agents that promote proliferation and neurogenesis are further evaluatedfor their ability to inhibit neurodegeneration and apoptosis of thehuman hippocampal multipotent stem cell-derived neurons.Neurodegeneration may be induced in culture using any number of inducersas known by those skilled in the art. Examples of neurodegenerativeinducers include: staurosporine, hypoxia reperfusion, free radicals,glutamate agonists, activated monocyte supernatant, beta amyloidpeptides and corticosteroids to name a few. Some of the neurogenicagents inhibit loss of neurons, inhibit caspase activation, inhibitnuclear fragmentation or condensation, inhibit lactate dehydrogenaseactivity among other effects on neurodegeneration. Using staurosporineas an example, 125 nM of a staurosporine solution is added along with 10μM of one of the listed neurogenic agents to human hippocampal stemcells differentiated for 3-4 weeks.

A number of neurogenic agents inhibit neuronal loss, caspase-3 activityand/or nuclear fragmentation compared to staurosporine vehicle control.These NSI small molecule agents are all effective at in vitroneurogenesis as determined by methods described. More particularly,human hippocampal stem cells differentiated for a period of 3-4 weeksare treated with an agent for 8-24 hrs in a neuronal maintenance mediaprior to treatment with an apoptotic or neurodegenerative insults suchas staurosporin, TNFα and LPS-stimulated monocyte supernatant and thelike. Following the treatment period, media is removed from the treatedcells and the cells are fixed and stained. Staining may include Hoechststain, MAP-2ab (AP-20) antibody staining, active caspase-3 antibody orother measures of neurodegeneration and apoptosis. Instrumentation suchas a Molecular Devices fluorescent plate reader or a Genomics Array ScanII instrument and the like may be used to measure the degeneration andapoptosis.

FIG. 5 illustrates the results of an assay for an in vitro model ofneuroinflammation. One secondary screen for neuroprotection being usedin one embodiment of the present invention includes an in vitro modelthat physiologically mimics stress. Since a large body of literaturesuggests that stress-induced consequences on cells are mediated bycytokines and other proinflammatory agents, cellular stress induced byneuroinflammation in culture is available as a reasonable approximationof the neuroinflammation process in human brains. Thus, a secondaryassay to measure the effect of a compound to protect neurons fromLPS-activated human monocyte supernatant is established.

As illustrated in FIGS. 5 and 6, the results of the assay include LDHrelease, which measures cell damage/death, and the actual cell countingof neurons. Human hippocampal stem cells are differentiated for fourweeks and then treated with the spent medium collected from humanmonocytes pre-treated for twenty-four hours with either the vehiclealone (RPMI medium) or with 0.5 ug/ml lipopolysaccharide (LPS) Lactatedehydrogenase (LDH) in the neuronal media was measured at 24 hours postincubation of the hippocampal cells with monocyte supernatant while thenumber of neurons indicated by MAP-2 staining was measured afterforty-eight hours of treatment. As illustrated in FIGS. 5 and 6, theresults of the assay include LDH release, which measures celldamage/death, and the actual cell counting of neurons.

In FIG. 6, of the sixteen compounds tested by this secondary assay, onecompound demonstrated protection measured by LDH release at 24 hourspost-treatment and seven showed neuroprotection measured by counting ofneurons at 48 hours post-treatment.

Method for Measuring Caspase-3 Activity as a Measure of Apoptosis

Another assay used to determine the ability of a compound to provideneuroprotection is an apoptosis assay. Apoptosis is measured using theactivity of an early matter of the programmed cell death pathway,caspase-3. Following 24 hours or 48 hours of apoptotic-induction in thepresence and absence of compound treatment, the media is removed fromthe wells, and 50 μl cell lysis buffer is added to each well, 50 μl of2× reaction buffer containing 10 mM DTT is then added to the lysate ineach well. The contents of two wells are combined (for example: rowsA/H, B/G, C/F, D/E), and 5 μl of substrate is added per well asrecommended in the protocol for a caspase-3 fluorometric assay kit(Biovision) known in the art. The plate is incubated at 37° C. for 60minutes. The contents of each well are then transferred to a freshplate, and the fluorescence is determined with an emission wavelength of400 nm and an excitation of 505 nm using a fluorescent plate reader suchas the Molecular Devices Gemini Fluorescent Plate Reader. Caspase-3activity is expressed as μM of cleaved substrate per hour. Substrateconcentration in μM is run for each plate to determine activity valuesfor each treatment condition.

FIGS. 7A, 7B and 7C illustrate an example of a neuroprotection profileof tested compounds against staurosporine-induced human hippocampalneurodegeneration measured by the remaining number of neurons, anincrease in nuclear fragmentation, and an increase in intracellularlevels of activated caspase-3/-7 detected by immunostaining,respectively. Human hippocampal stem cells that have been differentiatedfor 3-4 weeks undergo a twenty-four hour treatment with 125 nMstaurosporine solution to cause neuronal injury and death. The effect ofthe compound treatment to inhibit the staurosporine-induced neuronalloss and injury is shown in each of FIGS. 7A, 7B and 7C. Each barrepresents n=6 wells of the mean+/−the SEM from a single experiment.

FIG. 8 is a bar graph illustrating an example of a neuroprotectionprofile of the compounds against apoptosis induced by beta amyloidpeptide 25-35, part of a larger peptide found in plaques in Alzheimer'sbrains, as a model of Alzheimer's disease. Caspase-3 activation plays acentral role in apoptosis and is an indicator of the occurrence ofapoptosis if detected. Three of the compounds, NSI-182, NSI-144 andNSI-130, are shown to inhibit apoptosis induced by beta amyloid peptide25-35. Bars represent mean±SD from five wells.

In Vivo Neurogenic Effects of NSI Compounds

In one embodiment, fifteen of the compounds from Table I above areadministered orally to mice at 10 mg/kg for ten days and co-administeredbromodeoxyuridine (BrdU) by intraperitoneal injection for the first sixdays. One class of compounds caused a significant increase inneurogenesis. This class, broadly defined as “isonicotinamides” and,more specifically of the structures, NSI-189 and NSI-158, wereespecially effective at promoting neurogenesis in the dentate gyrus ofC57B16 adult mice following ten-day consecutive oral administration. Forexample, in FIGS. 9A and 9B, BrdU immunostainings of hippocampal slicesfrom two mice treated with either vehicle alone or NSI-158 areillustrated. In FIGS. 9A and 9B, the mice are treated daily for ten dayswith vehicle alone (FIG. 9A) or orally with various test compounds at 10mg/kg (FIG. 9B). The animals are injected daily with BrdU for the firstseven days. At the end of the ten-day period, the animals are perfusedand their brains sliced for immunostaining with anti-BrdU antibody andanalysis. FIG. 9B illustrates that the NSI-158-treated brain has fargreater dividing cells in the dentate gyrus of the hippocampus than thevehicle treated mouse illustrated in FIG. 9A. Therefore, it is apparentthat, compared to the vehicle treatment, the NSI-158 treatment caused anincrease in dividing cells in the dentate gyrus, especially in thegranular cell layer which is made up of only neuronal cells.

In FIGS. 10A and 10B, seven compounds are demonstrated to be especiallyeffective in increasing neurogenesis in the dentate gyrus of healthyadult mice. Mice were treated with the various test compounds for invivo neurogenesis in two cohorts as described above. The cells from onedentate gyrus of each mouse in each test group were counted and averageBrdU immunoreactivity values are calculated. The post-mortem cell countsof BrdU-immunopositive cells of the two cohorts representing an absolutenumber of dividing cells indicate increased neurogenesis as shown inFIGS. 10A and 10B. The symbols, * and ** indicate immunoreactivityincreases of statistically significant p values.

The ability to promote in vivo neurogenesis using these orallyadministered compounds suggest that these agents could be beneficial fora number of disease states including depression, aging, stroke,Alzheimer's disease and mild cognitive impairment, to name a few. Withthe production of new neurons in the granular cell layer these samecompounds could promote cognitive enhancement to counteract loss ofneurons due to disease, injury or age. These could be useful whencognition enhancement in humans might be beneficial for military andother purposes. It should be appreciated that other potential uses arecontemplated herein and that this list includes only some of thepotential uses for which these neurogenic agents appear ideally suited.

Further Studies of NSI-Compound Effects on the Stages of In VitroNeurogenesis

In another in vitro assay, compounds of the fused imidazole,iminopyrimidine, nicotinamide, aminomethyl phenoxypiperidine andaryloxypiperidine types are evaluated for their effect on thedifferentiation of the culture-born cells to mature neurons. Moreparticularly, multipotent human hippocampal stem cells are treated inthree doses over a one-week period in N2b media and in the presence ofan agent of the type mentioned above. The cells are then fed twiceweekly for approximately two more weeks in N2b or Neuralbasal+B27 Media(Gibco) and then cells are fixed using 4% paraformaldehyde. Cells arewashed 3× with PBS (pH 7.4) and then stained using a nuclear stain(Hoechst or DAPI) and a mature neuronal marker, MAP-2ab (AP-20)antibody. A secondary antibody, Alexa Fluor 488 labeled goat anti-mouseIgG, is used to identify by fluorescent labeling the mature neurons. Thefluorescent intensity and number of cells labeled in a multi-plateformat can be measured using for instance a Array Scan II (Cellomics) orsimilar instrumentation.

Effects of the long-term NSI compound treatment on human hippocampalstem cells in the absence of mitogen is illustrated in FIG. 11. In FIG.11, differentiating hippocampal human stem cells are treated withvehicle alone or with the test compounds for three weeks. Increase incell number was determined compared to the control. Bars representmean+/−SD from six wells. FIG. 11 demonstrates that an increase inneuronal number with two weeks of treatment (5× with 10 μM) occursbeyond that observed previously with just one week of treatment with anNSI compound. Several examples of NSI compounds are shown to beeffective in continued neurogenesis. For example, NSI-130 and NSI-144 inaddition to being particularly effective in inhibiting apoptosis due tobeta amyloid as discussed above, are also effective in stimulatingfurther increases in neuron numbers over a relatively long time periodwhich may be advantageous for agents that are useful in chronic diseasessuch as Alzheimer's disease. These agents comprise a class of compounds,aminopyrimidines, that, as shown previously, inhibited apoptosis.Accordingly, the aminopyrimidine class of compounds appear ideallysuited for neurodegenerative long-term diseases including Alzheimer'sdisease and aging. Neurogenic agents that are neuroprotective like thesixteen compounds identified above might also be effective therapeuticsfor other neurodegenerative diseases in mammals including, but notlimited to, stroke, traumatic brain injury, Parkinson's disease, andmild cognitive impairment.

In FIG. 12 the exact number of dividing cells and neurons are determinedto confirm that the increase in neurons is due to proliferation followedby differentiation. In FIG. 12, hippocampal stem cells are plated in a96-well plate. The cells are then treated with one of the agents listedalong with a two-day pulse of bromodeoxyuridine which is taken up intodividing cells thereby labeling mitotic cells with BrdU. After threeweeks of continuous treatment with the listed agents, the cells arefixed and stained for BrdU immunoreactivity. BrdU-positive cells aredouble-stained with the marker of mature neurons, anti-MAP-2 to identifycells that have differentiated into mature neurons. The BrdU+/MAP-2ab+co-stained cells which have differentiated into mature neurons aredistinguished from control BrdU-immunostained cells which areproliferating stem cells. A number of agents, especially NSI-127,NSI-144, NSI-183 and NSI-190 appear to induce proliferation and thendifferentiation into neurons into the second and third week followingthe removal of stem cell mitogen.

As illustrated in FIG. 13, one NSI compound provided as a continuoustreatment for ten days further increases the number of dividing cellscompared to the seven-day treatment suggesting that the proliferationphase of neurogenesis continues past the first week. The results of theassay further suggest that the proliferation involves a committedneuronal progenitor. In FIG. 13, mitotic cells are labeled with BrdUduring the last forty-eight hours of a two-week treatment with the testcompounds. BrdU alone and BrdU together with TuJ1 as co-stained cellsare quantified.

As described in pending U.S. patent application Ser. No. 10/728,652,leukemia inhibitory factor (LIF), a cytokine growth factor, was selectedfrom among several known neurotrophic factors as a positive controlagainst which tested compounds can be compared. The selection of LIF asa positive control was based on its properties to increase the number ofneurons and glia by two to three-fold. This effect is both consistentwith the neural stem cell system in which the cells respondappropriately to the positive control by enhanced differentiation and/ormitosis, and achieves the objective of the assay method in which suchcellular responses can be measured reproducibly and quantifiably. Thus,the use of LIF in FIG. 13 is as a positive control to discriminatelytest agents for selectively possessing neurogenic activity.

Results from NSI-106 and the positive control, LIF treatment are shownas an example. Unlike with LIF, NSI-106 produces mostly co-stainedcells, indicating that the mitotic population is committed neuronalprogenitors. The bars represent the mean±SD from each of six wells.

FIG. 14 illustrates committed neuronal progenitor production by NSI-106.Differentiating hippocampal stem cells are treated with NSI-106 for 10days, fixed, and stained for Ki-67 protein to detect dividing cells aswell as TuJ1 to detect cells that have differentiated into neurons,Ki-67 protein is an endogenous marker of mitosis. As illustrated in FIG.14, significant number of cells are co-stained as indicated by Ki-67positive (blue nuclei) and TuJ1 positive (strongly green) cellsdemonstrating neuronal morphology. This observation suggests thatNSI-106 not only increases the number of dividing stem cells but causesan increase in dividing neuronal precursors or committed neuronalprogenitors.

In addition, referring back to FIG. 8, possible neuronal progenitorsinclude cells with neuronal morphology seen with Ki-67 positive and TuJ1positive in response to NSI-106 treatment after one week of humanhippocampal stem cells in the absence of mitogen.

The compound NSI-106 and similar agents could be useful especially in amore lipophilic form, as understood by those skilled in the art topromote division of neuronal progenitors in brain. This agent could beuseful for a number of neurological indications. This agent and thoselike, it even in its present form, could be useful with any endogenousor transplanted stem cell to promote a neurogenic phenotype. Theneurogenic phenotype could then be used for purposes of reversing orimproving any number of neurological indications, for example, thisagent could be given in utero or early postnatally for the purpose ofimproving brain or spinal nerve development. This compound might also beuseful in stimulating embryonic stem cells to produce a neuronalprogenitor that could be differentiated into neurons. Other uses of sucha compound include hippocampal replenishment of neurons replacing,replenishing or enhancing any neuronal phenotype in brain, spinal cordand peripheral nervous system. This compound could then be useful fornot only CNS indications but any nerve cell disease or injury.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

TABLE I NSI # CLogP Structure 106 3.166

127

130 3.12

142

143 1.44

144

149

150

157

158

182 3.01

183

185

189 4.497

190 4.32

137

TABLE II Stauro % Inhib Neuron Neuron Neuron Long Term Chemical NumberRatio % Neuron Number Treatment Descriptors % Prolif (% of (% of RatioNeuron# Nuclear Frag 2° Assay Prolif/#Neur/ NSI # LBB (Alamar) Control)Control) EC50/Eff EC50/Eff Caspase-3 Cell#/Apop Neur Ratio 106 −1.1 CNS(−) 211 ± 48  77 ± 12  92 ± 6 0.3 nM, r²0.59 0.07 nM, r²0.75 16 ± 19 92± 3 165% @100 nM 157% @300 nM N 137 ± 8  N 120 ± 5  127 −1.0 CNS (−) 181± 14  81 ± 13 104 ± 8 0.1 nM, r²0.65 1.5 nM, r²0.5 N 103 ± 8  114% @10nM 137% @30000 nM N 135 ± 21 N 131 ± 15 130 −1.5 CNS (−)  92 ± 33 124 ±8  128 ± 4 0.1 nM, r²0.39 2164 nM, r²0.70 N 115 ± 11 144% @30,000 nM202% @10000 nM 56 ± 17 128 ± 30  92 ± 12 142 0.1 CNS (++) 115 ± 13 152 ±22  125 ± 15 0.07 nM, r²065 0.001 nM, r²0.70 N 105 ± 26 123% @30,000 nM175% @30000 nM N 146 ± 34 120 ± 21 143 0.01 CNS (++)  97 ± 16 143 ± 6  130 ± 14 445 nM, r20.82 4.8 nM, r2 0.62 N 153 ± 33 112% @30000 nM 115%@30000 nM 123 ± 77  102 ± 14 103 ± 12 144 −0.8 CNS (−) 149 ± 15 179 ± 29137 ± 8 18.1 nM, r²0.73 13.5 nM, r²0.96 N  97 ± 21 138% @300 nM 186%@300 nM N 170 ± 46 N 123 ± 12 149 −0.3 CNS (+) 216 ± 28 113 ± 16 100 ± 6333 nM, r²0.75 <0.1 nM, r²0.66 47 ± 15  89 ± 13 111% @1000 nM 124% @1000nM N 171 ± 19 N 143 ± 11 150 −0.2 CNS (+) 177 ± 44 107 ± 29  99 ± 6 <.01nM, r²0.77 85 nM, r²0.90 110 ± 25 112% @30000 nM 227% @3000 nM 120 ± 10105 ± 7  157 −0.8 CNS (−)  218 ± 140 135 ± 15 142 ± 6 >1000 nM,r²0.67 >1000 nM, r²0.75 N 146 ± 19 146% @30000 nM 113% @10000 nM N 139 ±43 Tox hi dose Tox hi dose N 107 ± 22 158 −0.5 CNS 236 ± 79  93 ± 17 106± 9 36 nM, r²0.48 0.2 nM, r²0.88 22 ± 18 99 ± 6 106% @3000 nM 145% @300nM N 141 ± 24 48 ± 14 114 ± 14 182 −0.5 CNS 74 ± 8 95 ± 9  125 ± 10 0.01nM, r²0.54 0.1 nM, r²0.83 N N/N 95 ± 6 112% @10000 nM 123% @300 nM 85 ±45 78 ± 34/ 121 ± 13 108 ± 21  100 ± 27 111 ± 13 183 −0.6 CNS  69 ± 18128 ± 13 131 ± 4 No Curve 0.05 nM, r²0.60 N N  99 ± 19 112% @1 nM 140%@300 nM N 40 ± 33 148 ± 30 N N 111 ± 16 185 −1.3 CNS (−)  73 ± 26 141 ±70  135 ± 15 >1000 nM, r²0.895 786 nM, r²0.538 30 ± 19/  83 ± 25 112%@30000 nM 132% 3000 nM 13 ± 12 155 ± 30 N/N 105 ± 19 189 0.3 CNS (++) 70 ± 21 102 ± 41  130 ± 19 411 nM, r²0.82 253 nM, r²0.76 N/N 144 ± 17141% @30000 nM 165% @30000 nM 121 ± 74/ 110 ± 26 182 ± 39  96 ± 19 1900.1 CNS (++)  76 ± 14 130 ± 22 127 ± 9 <.001 nM, r²0.36 13 nM, r²0.64 42± 25/  98 ± 11 128% @1 nM 165% @300 nM 32 ± 1 121 ± 19 42 ± 30/N 100 ±12 137 −0.85 CNS (−) 108 ± 28 151 ± 40 116 ± 8 >1000, r²0.77 >1000 nM,r²0.75 N 111 ± 10 126% @30000 nM 145% @30000 nM 87 ± 41 139 ± 35 N  98 ±18

The invention claimed is:
 1. A composition comprising a neurogenicagent, said neurogenic agent including a compound having the structureselected from the group consisting of

and the pharmaceutically acceptable salts thereof.
 2. A compound havingthe structure

or the pharmaceutically acceptable salts thereof.
 3. A pharmaceuticalcomposition comprising the compound of claim 2, along with apharmaceutically acceptable carrier.
 4. A method for inhibiting neuronaldeath or for inducing proliferation and differentiation of a neuronalprogenitor cell into a neuron, which method comprises contacting saidneuron or cell with an effective amount of a composition containing asactive ingredient a compound of claim
 2. 5. A method of increasing thenumber of neurons in a subject which method comprises administering tosaid subject an effective amount of a composition containing as activeingredient a compound of claim
 2. 6. The method of claim 5, wherein thesubject is an adult human.
 7. The method of claim 5, wherein thecomposition is administered orally.
 8. The method of claim 5, whereinthe composition is administered in combination with at least one stemcell.
 9. The method of claim 8, wherein the stem cell is multipotential.10. The method of claim 8, wherein the stem cell is isolated from thehippocampus.
 11. The method of claim 10, wherein the stem cell isisolated from the dentate gyros of the hippocampus.
 12. The method ofclaim 8, wherein the stem cell is genetically modified to enhance themitotic capacity of the cell.
 13. The method of claim 12, wherein thestem cell is genetically modified to over-express c-myc in response to aconditional activation system.
 14. The method of claim 8, wherein thestem cell is differentiated prior to administration to the subject.